PREPARING CULTURE PLATES (not necessary for feeder-dependent cells)
Pipet sufficient Gelatin (0.1%) to cover bottom of the plate. Set in hood for 5 minutes. Aspirate off gelatin.
THAWING AND CULTURING ES CELLS
Day 1 REMOVE DMSO
1) Thaw cells quickly in 37degree bath, hold and check every 15 sec or so until the chunk slides.
2) With a 1ml pipet suck up melted cells and SLOWLY add to 9ml of media.
3) Suck up 1-2ml of mixture, add to ice chunk, then suck up from cryovial SLOWLY.
4) Spin tube in centrifuge (setting 3 for 4 min, 210g, 500rpm).
5) Aspirate Media from pellet (no need to touch pellet, tilt tube toward tip).
PLATE
1) Resuspend pellet in 2-3ml of media
2) Add cells to a 10cm plate pre-filled with 6ml of media (for 1 x 10^4 cells), swirl then move back and forth
3) Place on top shelf of incubator (check temp and H20)
Day 2: Monitor cells and rinse with 1XPBS, replace media to remove floaters.
Day 3: Feed, rinse in 1X PBS if necessary
Day 4: Feed, Recover 3-4 hrs, Split 1:2 or 1:3
Day 5: Feed
Day 6: Feed and electroporate or split 1:2
24 hours after electroporation, treat with G418 (100X stock= 180mg/ml)
Feed and treat for 8-12 days
Pick colonies on days 8-12
PASSAGING CELLS
1) Rinse cells with PBS.
2) Incubate with warmed Trypsin (Trypsin .25%, Versene .02%) for 10-15 min. at 37 degrees with occasional agitation
3) Add equal volumes of media to stop Trypsin and pipet vigorously 10-20X, check cells under the microscope to see if they have dissociated well.
4) Add to gelatinized dishes filled with media. Swirl then move back and forth.
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Pipet sufficient Gelatin (0.1%) to cover bottom of the plate. Set in hood for 5 minutes. Aspirate off gelatin.
THAWING AND CULTURING ES CELLS
Day 1 REMOVE DMSO
1) Thaw cells quickly in 37degree bath, hold and check every 15 sec or so until the chunk slides.
2) With a 1ml pipet suck up melted cells and SLOWLY add to 9ml of media.
3) Suck up 1-2ml of mixture, add to ice chunk, then suck up from cryovial SLOWLY.
4) Spin tube in centrifuge (setting 3 for 4 min, 210g, 500rpm).
5) Aspirate Media from pellet (no need to touch pellet, tilt tube toward tip).
PLATE
1) Resuspend pellet in 2-3ml of media
2) Add cells to a 10cm plate pre-filled with 6ml of media (for 1 x 10^4 cells), swirl then move back and forth
3) Place on top shelf of incubator (check temp and H20)
Day 2: Monitor cells and rinse with 1XPBS, replace media to remove floaters.
Day 3: Feed, rinse in 1X PBS if necessary
Day 4: Feed, Recover 3-4 hrs, Split 1:2 or 1:3
Day 5: Feed
Day 6: Feed and electroporate or split 1:2
24 hours after electroporation, treat with G418 (100X stock= 180mg/ml)
Feed and treat for 8-12 days
Pick colonies on days 8-12
PASSAGING CELLS
1) Rinse cells with PBS.
2) Incubate with warmed Trypsin (Trypsin .25%, Versene .02%) for 10-15 min. at 37 degrees with occasional agitation
3) Add equal volumes of media to stop Trypsin and pipet vigorously 10-20X, check cells under the microscope to see if they have dissociated well.
4) Add to gelatinized dishes filled with media. Swirl then move back and forth.
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